Adaptable tissue-specific endothelial cells for organ regeneration

项目摘要 我们提议的研究计划的首要目标是开发一条发现管道，使 治疗性工程组织特异性内皮细胞转录密码子的鉴定 心脏、肺和血液的器官再生。器官再生疗法承诺无限制地获得 换掉的纸巾。然而，尽管在揭示器官的分子基础方面取得了突破 形态发生学和器官学技术，再生医学向临床转化面临 障碍。这些瓶颈在一定程度上是由于对壁龛细胞如何协调器官缺乏了解 修理。具体地说，提供再生信号的血管壁龛细胞的贡献尚未实现。 这个R35应用程序建立在一个新的命题上，即器官损伤后愈合不良是由于 组织特异性内皮细胞的功能障碍和丢失。这一纲领性提案检验了这样一种假设： 受损器官中干细胞的重建依赖于提供的促再生血管分泌信号 通过组织特异性血管壁龛内皮细胞。我们已经证明，通过部署定义的血管分泌，器官型内皮细胞 支持肺、心、肝和造血再生的因素。因此，欧洲共同体作为动态的、 组织特定的小生境细胞对组织的动态平衡和修复至关重要。为了测试这一点，并为 在治疗方面，我们已经通过转导小鼠、非人灵长类和人类内皮细胞来改造出适应性强的内皮细胞。 成体成熟内皮细胞转导因子ETV2与人诱导多能分化 干细胞(IPSCs)转化为通用的类胎儿内皮细胞(IVECs)，可告知诱导 器官型TF。这些适应性的iVECs和R-VECs将与心脏、肺和血液器官共同培养 在体外或体内输注进行器官修复的小鼠，以确定在这些器官中诱导器官型转录因子的情况 细胞。培养的iVECs和R-VECs将被回收并接受RNA图谱和从头开始的基序 鉴定诱导组织特异性转铁蛋白的发现(S)。被识别的TF将被过度表达或被删除 在ECs中，验证它们在维持器官类型和血管分泌特征以进行器官修复方面的功能。我们期待着 器官型内皮细胞移植将促进持久的组织修复，而不会引发肿瘤形成 或纤维化症。我们已经启动了FDA批准的人体临床试验，以检查 异基因非专利EC输注用于造血恢复。作为后续行动，我们打算评估这一贡献 来源于非人灵长类动物的R-VECs或iVECs-在有 意在将器官型内皮细胞的潜力转化为临床。拟议方案的预期结果 研究是鉴定组织特异性血管和血管的分子信号和转录决定因素 血管分泌异质性。该计划的目标与NHLBI R35奖的使命相吻合，以开发创新 再生发现管道促进安全有效的心、肺和血液治疗 疾病。

PROJECT ABSTRACT The overarching goal of our proposed research program is to develop a discovery pipeline that will enable identification of transcriptional codes for engineering tissue-specific endothelial cells (ECs) for therapeutic organ regeneration of heart, lung and blood. Therapies for organ regeneration promises unlimited access to the replacement tissues. However, despite breakthroughs in uncovering the molecular underpinnings of organ morphogenesis and organoid technology, translation of regenerative medicine to the clinic has confronted with hurdles. These bottlenecks are in part due to the lack of understanding as to how niche cells coordinate organ repair. Specifically, contribution of vascular niche cells that supply regenerative signals has not been realized. This R35 application builds upon the novel proposition that poor healing after organ damage is due to the dysfunction and loss of the tissue-specific ECs. This programmatic proposal examines the hypothesis that reconstitution of stem cells in injured organs is dependent on the pro-regenerative angiocrine signals supplied by tissue-specific vascular niche ECs. We have shown that organotypic ECs by deploying defined angiocrine factors support lung, cardiac, hepatic and hematopoietic regeneration. Thus, ECs perform actively as dynamic, tissue-specified niche cells critical for tissue homeostasis and repair. To test this and to set up the stage for therapies, we have engineered adaptable mouse, nonhuman primate and human ECs by transducing the transduction factor (TF) ETV2 into adult mature ECs (R-VECs) and differentiating human induced pluripotent stem cells (iPSCs) into generic fetal-like ECs (iVECs) that could inform on the pathways that induce organotypic TFs. These adaptive iVECs and R-VECs will be cocultured with heart, lung, and blood organoids in vitro or infused in vivo in mice undergoing organ repair to identify the induction of organotypic TFs in these cells. The educated iVECs and R-VECs will be recovered and subjected to RNA profiling and de novo motif discovery to identify induced tissue-specific TF(s). The identified TFs will be overexpressed or knocked down in ECs, to validate their function in sustaining organotypic and angiocrine profile for organ repair. We anticipate that transplantation of organotypic ECs will promote long-lasting tissue repair without provoking tumorigenesis or fibrosis. We have initiated FDA-approved human clinical trials to examine the safety and efficacy of allogeneic generic EC infusion for hematopoietic recovery. As a follow up, we intend to assess the contribution of R-VECs or iVECs-derived from nonhuman primates to regeneration in the pigtail macaque monkeys with the intention of translating the potential of organotypic ECs to clinic. The expected outcomes of the proposed research are identification of molecular signals and transcriptional determinants of tissue-specific vascular and angiocrine heterogeneity. Goals of this proposal fit with the mission of NHLBI R35 award to develop innovative regenerative discovery pipeline to promote safe and efficacious treatments for cardiac, pulmonary and blood maladies.